Soluble FasR ligand-binding domain: high-yield production of active fusion and non-fusion recombinant proteins using the baculovirus/insect cell system.
We used the recombinant baculovirus/insect cell system to express two soluble forms of the mouse Fas receptor (mFasR) extracellular domain (ECD): a monomer comprising the entire ligand-binding portion of mFasR followed by a carboxy-terminal hexa-histidine extension aiding purification by immobilized metal affinity chromatography and an immunoadhesin in which the same 148 residues were fused to the Fc portion of a truncated human IgG1 immunoglobulin heavy chain. Both constructs harboured a 24 base pairs insertion placed upstream of the initiating ATG [Peakman, Charles, Sydenham, Gewert, Page, and Makoff (1992) Nucleic Acids Res. 20, 6111-6112]. Despite its hexa-histidine extension, the monovalent recombinant protein from crude culture media failed to bind immobilized Ni2+ unless proteins were first precipitated twice by ammonium sulphate. The overall procedure then yielded approximately 10mg/l of protein which could be purified to near homogeneity using two additional chromatographic steps. The glycosylated polypeptide migrated as a band of Mr=(21-31) x 10(3) in SDS/PAGE and was monomeric in physiological buffers. Under non-reducing conditions, denaturation in 6 M guanidinium chloride was reversible after slow removal of the denaturing agent. The mFasR immunoadhesin was secreted (approximately 5-10 mg/l) as a disulphide-linked homodimer, and endowed with ligand-binding activity since it could bind FasL on the surface of D11S, FasL-expressing cells. When tested for their ability to inhibit FasR-dependent cell lysis, the soluble dimeric immunoadhesin markedly inhibited FasL-mediated cytotoxicity (IC50 approximately 30 nM), and was approximately 6 times as effective as its monomeric counterpart.
Mahiou
J
J
INSERM U 373, Faculté de Médecine Necker, 156 rue de Vaugirard, 75730 Paris Cedex 15, France.
Abastado
J P
JP
Cabanie
L
L
Godeau
F
F
D000818:Animals; D000954:Antigens, Surface / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D017209:Apoptosis; D016367:Baculoviridae; D002846:Chromatography, Affinity; D019281:Dimerization; D053222:Fas Ligand Protein; D006801:Humans; D008024:Ligands; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012995:Solubility; D018411:Spodoptera; D019014:fas Receptor / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
D000954:Antigens, Surface; C505661:FASLG protein, human; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D008024:Ligands; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D019014:fas Receptor